Structure-activity relationships of uridine 5′-diphosphate analogues at the human P2Y6 receptor

Article abstract of DOI:10.1021/jm060485n

The structure-activity relationships and molecular modeling of the uracil nucleotide activated P2Y₆ receptor have been studied. Uridine 5′-diphosphate (UDP) analogues bearing substitutions of the ribose moiety, the uracil ring, and the diphosph

Full text of DOI:10.1021/jm060485n

5532
J. Med. Chem. 2006, 49, 5532-5543
Structure-Activity Relationships of Uridine 5′-Diphosphate Analogues at the Human P2Y6
Receptor
Pedro Besada,† Dae Hong Shin,† Stefano Costanzi,‡ Hyojin Ko,† Christophe Mathe´,§ Julien Gagneron,§ Gilles Gosselin,§
Savitri Maddileti,# T. Kendall Harden,# and Kenneth A. Jacobson*,†
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and DigestiVe and Kidney Diseases,
National Institutes of Health, Bethesda, Maryland 20892, Computational Chemistry Core Laboratory, National Institute of Diabetes and
DigestiVe and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, Laboratoire de Chimie Organique Biomole´culaire
de Synthese, UMR 5625 CNRS-UM II, UniVersite´ Montpellier II, Place E. Bataillon, 34095 Montpellier, Cedex 5, France, and
Department of Pharmacology, UniVersity of North Carolina School of Medicine, Chapel Hill, North Carolina 27599
ReceiVed April 25, 2006
The structure-activity relationships and molecular modeling of the uracil nucleotide activated P2Y6 receptor
have been studied. Uridine 5′-diphosphate (UDP) analogues bearing substitutions of the ribose moiety, the
uracil ring, and the diphosphate group were synthesized and assayed for activity at the human P2Y6 receptor.
The uracil ring was modified at the 4 position, with the synthesis of 4-substituted-thiouridine 5′-diphosphate
analogues, as well as at positions 2, 3, and 5. The effect of modifications at the level of the phosphate chain
was studied by preparing a cyclic 3′,5′-diphosphate analogue, a 3′-diphosphate analogue, and several
dinucleotide diphosphates. 5-Iodo-UDP 32 (EC50 ) 0.15 µM) was equipotent to UDP, while substitutions
of the 2′-hydroxyl (amino, azido) greatly reduce potency. The 2- and 4-thio analogues, 20 and 21, respectively,
were also relatively potent in comparison to UDP. However, most other modifications greatly reduced potency.
Molecular modeling indicates that the â-phosphate of 5′-UDP and analogues is essential for the establishment
of electrostatic interactions with two of the three conserved cationic residues of the receptor. Among
4-thioether derivatives, a 4-ethylthio analogue 23 displayed an EC50 of 0.28 µM, indicative of favorable
interactions predicted for a small 4-alkylthio moiety with the aromatic ring of Y33 in TM1. The activity of
analogue 19 in which the ribose was substituted with a 2-oxabicyclohexane ring in a rigid (S)-conformation
(P ) 126°, 1′-exo) was consistent with molecular modeling. These results provide a better understanding of
molecular recognition at the P2Y6 receptor and will be helpful in designing selective and potent P2Y6 receptor
ligands.
Introduction
by UTP, the P2Y14 by uridine 5′-diphosphate glucose, and the
P2Y6 receptor by uridine 5′-diphosphate (UDP, 1, Chart 1).16
P2 nucleotide receptors consist of two families: G-protein-
coupled receptors (GPCRs) designated P2Y, which include eight
subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11-P2Y14), and ligand-
gated cation channels designated P2X, which include seven
subtypes (P2X1-P2X7). All of these subtypes have been cloned
and functionally characterized.1-10 The family of P2Y receptors
can be further divided in two different subgroups based on
overall sequence similarity, coupling to specific G proteins, and
second messenger responses. The P2Y1-like subgroup (P2Y1,
P2Y2, P2Y4, P2Y6, P2Y11) is Gq-coupled and stimulates
phospholipase C (PLC), and the P2Y12-like subgroup (P2Y12,
P2Y13, P2Y14) is Gi-coupled and inhibits adenylyl cyclase
(AC).11 The distribution of P2Y receptors is broad, and certain
of these are of therapeutic interest including antithrombotic
therapy, modulation of the immune system and cardiovascular
system, inflammation, pain, diabetes, and treatment of cystic
fibrosis and other pulmonary diseases.12-15 Whereas P2X
receptors tend to be activated principally by adenine nucleotides,
P2Y receptors are activated by adenine and/or uracil nucleotides.
The P2Y2 receptor is activated by both uridine 5′-triphosphate
(UTP) and adenosine 5′-triphosphate (ATP), the P2Y4 receptor
The P2Y6 receptor is distributed in various tissues including
lung, heart, thymus, aorta, bone, spleen, digestive tract, placenta,
and brain. This receptor has been implicated in protection against
apoptosis induced by TNFR,17 enhancement of osteoclast
survival through NF-κB activation,18 regulation of electrolyte
transport in the airways,19 production of proinflammatory
cytokines and chemokines,20,21 and growth and contraction of
vascular muscles.22
The SAR (structure-activity relationship) of nucleotides in
activating the human P2Y6 receptor has been probed (Chart 1).
5-Br-UDP 2, UDP-â-S 3, and the dinucleotide triphosphate
INS48823 (P1-((2-benzyl-1,3-dioxolo-4-yl)uridine 5′) P3-(uri-
dine 5′) triphosphate) 4 are potent and/or stable agonists of the
P2Y6 receptor.5,18,19,22 We have reported that the South (S)
conformation of the ribose moiety is preferred for ligand
recognition by the P2Y6 receptor.23,35 The conformationally
constrained (N)-methanocarba (mc) derivative 5 was inactive,
and the (S)-methanocarba derivative 6 was moderately potent
in activating the P2Y6 receptor. Replacement of the uracil moiety
with other nucleobases 7-10 greatly reduced the potency at
the human P2Y6 receptor.24 To investigate more extensively the
SAR at the P2Y6 receptor, we synthesized a series of UDP
analogues, where modifications at the level of the uracil moiety,
ribose ring, and/or phosphate chain have been introduced.
Molecular modeling was carried out to predict sites of interaction
of the ligands with the P2Y6 receptor and to provide hypotheses
for the design of additional analogues.
* To whom correspondence should be addressed. Phone: 301-496-9024.
Fax: 301-480-8422. E-mail: kajacobs@helix.nih.gov.
† Laboratory of Bioorganic Chemistry, National Institute of Diabetes and
Digestive and Kidney Diseases.
‡ Computational Chemistry Core Laboratory, National Institute of
Diabetes and Digestive and Kidney Diseases.
§ Universite´ Montpellier II.
# University of North Carolina School of Medicine.
10.1021/jm060485n CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/04/2006

Uridine 5′-Diphosphate Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5533
Chart 1. Structures of UDP and Various Analogues and the Reported EC50 Values in Stimulation of PLC through the Recombinant
P2Y6 Receptor (Human, Unless Noted)16,22-24,35,a
a The EC50 for INS48823 is similar to that for UDP.18
To facilitate the comparison among receptors, throughout this
paper we use the GPCR residue indexing system, as explained
in detail elsewhere.11
combined with 31P NMR and HRMS indicated that for 17 the
diphosphate group is in the 3′ position. The synthesis of 2′-
deoxy-2′-aminouridine 5′-diphosphate 16 was accomplished
after the acylation of the amino group in 40 with a trifluoroacetyl
group to obtain 41. Phosphorylation of the 2′-trifluoroacetyl-
amino derivative 41 followed by an in situ deprotection of the
2′-amine provided the 5′-diphosphate analogue 16. Compound
16 was also obtained by reduction of the 2′-deoxy-2′-azidou-
ridine 5′-diphosphate 15 with H2 and Pd/C. The NMR spectrum
for 16 shows peaks at 4.61 ppm (H-3′) and 4.24, 4.16 ppm
(H-5′) in accordance with other 5′-diphosphates derivatives. The
preparation of compounds 28, 30, and 32 was accomplished by
another method, i.e., activation of the 5′-monophosphate by
reaction with 1,1′-carbonyldiimidazole followed by the addition
of phosphoric acid tributylammonium salt23 to provide the
corresponding diphosphate (Schemes 4 and 5).
The synthesis of various 4-substituted-thiouridine nucleosides
and nucleotides from uridine was described previously,27-29 and
various methods of S-alkylation were applied to a purine
nucleoside, 2-thioadenosine.26,30 By adapting these alkylation
procedures, we have developed a more direct synthesis of
4-substituted-thiouridine nucleotides starting with the com-
mercially available 4-thiouridine 5′-diphosphate (4-thio-UDP)
21 that allowed us to obtain the final compounds in only two
steps (Scheme 3).
The treatment of 4-thio-UDP with 0.25 M NaOH in methanol
for 2 h at room temperature gave the corresponding sodium
thiolate salt, which appeared as a yellow solid after the solvent
was lyophilized. The nucleotide diphosphate sodium salt was
selectively S-alkylated by reacting with an excess of the
corresponding alkyl halide at room temperature and using
dimethylformamide as solvent. Compounds 22-27 and 29 were
obtained following this procedure. Because of stability problems,
the ester derivatives 45 and 46 were directly subjected to
hydrolysis without purification by treatment with 0.25 M NaOH
at room temperature to afford the corresponding acid derivatives
26 and 29, respectively.
Results
Chemical Synthesis. Analogues of UDP 1 with modifications
in the ribose ring, uracil moiety, and phosphate chain, as well
as dinucleotides (Table 1), were synthesized. The ribose ring
was modified by removal of the hydroxyl group at the 3′
position, 13, replacement of the hydroxyl at the 2′ position with
various functional groups, 15-18, and with the substitution of
a 2-oxabicyclohexane ring (2-OBH) in place of the ribose ring,25
which fixed the sugar moiety in a rigid (S)-conformation, 19.
The uracil ring was modified at the 2 and 4 positions, with the
synthesis of 2-thiouridine diphosphate 20 and 4-substituted-
thiouridine diphosphate analogues 22-30, and at the 3 and 5
positions with 31 and 32, respectively. Finally, the synthesis of
2′-deoxy-2′-aminouridine 3′-diphosphate 17, the cyclic 3′,5′-
diphosphate 33, and the dinucleotides 34-37 as byproducts has
allowed us to study modifications at the level of the phosphate
chain. All the nucleotide analogues were prepared in their
ammonium or triethylammonium salt forms according to the
methods shown in Schemes 1-5 and tested in functional assays
of the P2Y6 receptor (Table 1). The nucleotide analogues were
characterized using nuclear magnetic resonance (1H, 13C, and
31P NMR and COSY) and high-resolution mass spectrometry.
The nucleotide diphosphates were obtained following two
different methods of phosphorylation. The one-pot method using
a sequential reaction with phosphorus oxychloride and phos-
phoric acid tributylammonium salt26 provided the 5′-diphosphate
analogues 13, 15, 16, 18-20 in moderate yields (Schemes 1
and 2). The phosphorylation of the 2′-amino-2′-deoxyuridine
40 using the one-pot method provided the 3′-diphosphate
1
derivative 17. The H NMR of compound 17 showed peaks at
5.01 ppm (H-3′) and 3.89 ppm (H-5′), whereas the chemical
shifts of 5′-diphosphate uridine analogues are usually at 4.65-
4.35 ppm (H-3′) and 4.40-4.15 ppm (H-5′). These results

5534 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18
Besada et al.
Table 1. In Vitro Pharmacological Data for UDP, 1, and Its Analogues in the Stimulation of PLC at Recombinant Human P2Y6 Receptors Expressed in
1321N1 Astrocytoma Cellsa
compd
modification
structure
EC50 at hP2Y6 receptor,b µM
1
11
()UDP)
()UMP)
0.013 ( 0.004
NEd
ribose-modified
12
13
14
15
16
17
18
2′-deoxy
3′-deoxy
()2′-deoxyuridine bisphosphate)
2′-deoxy-2′-azido
R7 ) H
1.72 ( 0.76
2.5 ( 0.8
NEd
1.5 ( 0.4
3.9 ( 0.9
NEd
R8 ) H
see structure above
R7 ) N3
2′-deoxy-2′-amino
R7 ) NH2
see structure above
R7 ) H, R6 ) F
e
2′-deoxy-2′-amino 3′-diphosphate
2′-fluoro-2′-deoxyara
(N)-methanocarba
5.5 ( 0.5
5
NEd
6
19
(S)-methanocarba-2′-deoxy
e
0.23 ( 0.05
3.5 ( 0.8
3′,4′-cyclopropylc
see structure above
uracil-modified
20
21
22
23
24
25
26
27
28
29
30
31
32f
2-thio
4-thio
4-methylthio
4-ethylthio
4-allylthio
4-benzylthio
4-carboxymethylthio
4-carboxamidomethylthio
4-carboxyethylthio
4-carboxypropylthio
4-hexylthio
R2 ) S
0.06 ( 0.01
0.08 ( 0.04
2.3 ( 0.6
R4 ) S
R4 ) SCH3
R4 ) SCH2CH3
0.28 ( 0.02
0.56 ( 0.09
0.80 ( 0.05
1.7 ( 0.3
R4 ) SCH2-CHdCH2
R4 ) SCH2C6H5
R4 ) SCH2CO2H
R4 ) SCH2CONH2
R4 ) S(CH2)2COOH
R4 ) S(CH2)3COOH
R4 ) S(CH2)5CH3
R3 ) CH3
18 ( 9
1.1 ( 0.2
8.8 ( 2.0
4.9 ( 0.2
3-methyl
5-iodo
3.3 (1.0
R5 ) I
0.015 ( 0.002
phosphate-and-ribose-modified
33
cyclic 3′,5′-diphosphate and 2′-ara-F
see structure above
NEd
dinucleotides
34
35
36
37
Up2U
R1 ) uridine-5′-
2.3 ( 0.9
U(4-ethylthio)p2U(4-ethylthio)
U(4-benzylthio)p2U(4-benzylthio)
Cp2C
R1 ) 4- SCH2CH3-uridine-5′-, R4 ) SCH2CH3
R1 ) 4- SCH2C6H5-uridine-5′-, R4 ) SCH2C6H5
R1 ) cytidine-5′-, R4 ) NH
<50% at 10 µM
<50% at 10 µM
NEd
a Unless otherwise noted, the groups are as follows: R1, R3, R5, R6 ) H; R2, R4 ) O; R7, R8 ) OH. b Agonist potencies were calculated using a
four-parameter logistic equation and the GraphPad software package (GraphPad, San Diego, CA). EC50 values (mean ( standard error) represent the
concentration at which 50% of the maximal effect is achieved. Relative efficacies (%) were determined by comparison with the effect produced by a
maximal effective concentration of reference agonist (UDP) in the same experiment. If no maximal effect is given, then 100% efficacy was achieved.
c Oxabicyclohexane ring system (2-OBH).25 d NE: no effect at 10 µM. e See Chart 1. f 32, MRS2693.
Nucleoside 5′-diphosphates are generally less stable than the
corresponding monophosphates. For this reason, for alkyl halides
in which heating is necessary for the alkylation reaction, the
starting material was the commercially available 4-thiouridine
5′-monophosphate (4-thio-UMP) 47 (Scheme 4). After the
formation of the sodium thiolate salt of the 4-thio-UMP, the
thio group of the uracil moiety was alkylated following the same
procedure as in Scheme 3. The alkylthio monophosphates
obtained were converted to the corresponding diphosphates
through a phosphorylimidazolide intermediate, which reacted
with the phosphoric acid tributylammonium salt.23 Compounds
28 and 30 were obtained following this procedure. The ester
derivatives 48 and 49, because of stability problems, were
directly subjected to the next reaction without purification.
The nucleotides were selectively S-alkylated using the
procedures described above. The 13C NMR spectrum of the
amide derivative 27 shows the peak corresponding to the
methylene of the acetamide at 33.0 ppm, typical of a carbon-
sulfur bond.
Pharmacological Activity. Activation of PLC by the nucle-
otide derivatives was studied in 1321N1 astrocytoma cells stably
expressing the human P2Y6 receptor.16,31 Removal of the 2′- or
3′-hydroxyl group of UDP led to a >100-fold decrease in
potency in 12 and 13, respectively. Substitution of the 2′-
hydroxyl group of UDP with an azido group and amino group
in 15 and 16, respectively, greatly reduced potency. The P2Y6
receptor is highly selective for 5′-diphosphate derivatives. Thus,
uridine 5′-monophosphate 11, 2′-deoxyuridine 3′,5′-bisphosphate
14,32 and a uridine 3′-diphosphate derivative 17 were inactive
as either agonists or antagonists.
Rigid ring systems also were used to determine the preferred
ribose conformation at the P2Y6 binding site. As previously

Uridine 5′-Diphosphate Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5535
Scheme 1. Preparation of Ribose- and Uracil-Modified UDP
the parent compound UDP. Among uncharged alkylated deriva-
tives of 21, the highest potency was observed with the S-ethyl
analogue 23, which was 20-fold less potent than UDP. In the
series of carboxyalkylthio ethers, the highest potency was
observed with the S-carboxyethylthio analogue 28. Substitution
at the 3 position in 31 markedly decreased potency, while
halogenation of the 5 position in the iodo analogue 32 resulted
in a molecule that was equal in potency to UDP. By analogy,
a 5-bromo analogue was previously reported to be roughly
equipotent to UDP.16
Analoguesa
The cyclic 3′,5′-diphosphate 33, which can be considered a
closely related cyclized analogue of bisphosphate 14, was
inactive at the P2Y6 receptor. The dinucleotide 34 was
moderately potent at the P2Y6 receptor. 4-Thioether modification
at both nucleobases in 35 and 36 reduced potency. A 4-O or
4-S was required for P2Y6 receptor activation, since the
corresponding NH analogue 37 was inactive.
Within a series of uracil dinucleotide 5′,5′-polyphosphates,
the triphosphates were the most potent and selective at the
human P2Y6 receptor. However, dinucleotide 5′,5′-diphosphates
also showed selectivity for the P2Y6 receptor.33,34 Therefore,
several dinucleotide diphosphates 34-37, including 4-alkylthio
analogues, were compared in this study. Only the simple Up2U
34 displayed substantial micromolar potency at the P2Y6
receptor.
a Reagents and conditions: (i) (1) POCl3, Proton Sponge, PO(OMe)3, 0
°C; (2) (Bu3NH+)2PO4H2-, Bu3N, DMF, 0 °C; (ii) CF3COOEt, DIEA, DMF,
room temp; (iii) H2, Pd/C, MeOH, room temp.
Scheme 2. Preparation of 2-OBH UDP Analoguea
Molecular Modeling. Docking studies of selected compounds
were conducted at the model of the membrane-embedded human
P2Y6 receptor.35 The binding mode of each studied compound
was obtained by subjecting the complex to two distinct
conformational searches, performed sequentially. These searches
were intended to exhaustively explore the possible docking poses
of the ligand by means of rotations and translations of the small
molecule within the binding pocket. Simultaneously, the
conformation of the residues lining the pocket was rearranged
to minimize the energy of interaction with the ligand.
a Reagents and conditions: (i) (1) POCl3, Proton Sponge, PO(OMe)3, 0
°C; (2) (Bu3NH+)2PO4H2-, Bu3N, DMF, 0 °C.
Scheme 3. Preparation of 4-Substituted-thio-UDP Analogues:
Procedure Ba
A schematic representation of the entire structure of the P2Y6
receptor complexed with 32 is given in Figure 1a, while Figure
1b shows the details of the receptor-ligand interactions. In
analogy with our previous P2Y6-UDP complex,35 the diphos-
phate moiety of 32 is coordinated by three cationic residues
from transmembrane domain 3 (TM3), TM6, and TM7, namely,
3.29, 6.55, and 7.39. The O at position 2 and the NH at position
3 of the uracil ring are H-bonded with S291(7.43) and Y33-
(1.33), respectively. Consistent with the equipotency of 32 and
UDP, the 5-iodo group does not show any sort of steric or
electronic incompatibility with the features of the P2Y6 binding
pocket. Figure 1c shows compound 6 docked into the putative
binding pocket of the P2Y6 receptor model. The locked
puckering of the pseudoribose ensures that the phosphate and
the nucleobase moiety of 6 maintain the optimal geometry for
the interaction with the P2Y6 binding pocket. The donation of
an H-bond from the NH at position 3 of the uracil ring to Y33-
(1.39), even though not essential for the recognition of the
nucleotides, apparently contributes to enhanced potency at the
P2Y6 receptor. Compound 31, which is incapable of donating
an H-bond to Y33(1.39) because of the methyl substituent at
the N3 position (Figure 1d), exhibited a 250-fold lower potency
than UDP. A decreased partial negative atomic charge on the
N3 might contribute to the decreased potency of 4-thio-UDP
(21) as a consequence of a weaker H-bond donation to Y33-
(1.39). Moreover, all the 4-thio substituted compounds, which
are incapable of donating an H-bond to Y33(1.39), exhibited
lower potency than the parent compound 4-thio-UDP (21).
Among these molecules, 4-ethylthio-UDP (23), 4-allylthio-UDP
a Reagents and conditions: (i) (1) 0.25 M NaOH, MeOH, room temp;
(2) RX, DMF, room temp; (ii) 0.25 M NaOH, H2O, room temp.
reported, a strong preference for the (S)-conformation was
indicated (compare 6 with 5). Another rigid ring system (2-
OBH) in 19 was tolerated at the P2Y6 binding site.
Among uracil-modified compounds, 2-thio-UDP 20 and
4-thio-UDP 21 were tested in order to probe the effect of the
electronegativity as well as the size of the exocyclic atoms at
positions 2 and 4. Both derivatives were 5-fold less potent than

5536 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18
Besada et al.
Scheme 4. Preparation of 4-Substituted-thio-UDP Analogues: Procedure Ca
a Reagents and conditions: (i) (1) 0.25 M NaOH, MeOH, room temp; (2) R1X, DMF, 90 °C; (ii) (1) 1,1′-carbonyldiimidazole, DMF, room temp; (2) Et3N
5% in 1/1 H2O/MeOH, room temp; (3) (Bu3NH+)2PO4H2-, Bu3N, DMF, room temp; (iii) 0.25 M NaOH, H2O, room temp.
Scheme 5. Preparation of 5-Halo and Phosphate- and Ribose-Modified UDP Analoguesa
a Reagents and conditions: (i) POCl3, Proton Sponge, PO(OMe)3, 0 °C; (ii) (1) 1,1′-carbonyldiimidazole, DMF, room temp; (2) Et3N 5% in MeOH, room
temp; (3) (Bu3NH+)2PO4H2-, Bu3N, DMF, room temp.
(24), and 4-benzylthio-UDP (25) showed the highest potency.
Molecular modeling results suggest that the hydrophobic
substituents at position 4 of the uracil ring establish favorable
interactions with the aromatic ring of Y33(1.39) and with the
hydrophobic pocket located between TM1 and TM2 (Figure 1e).
The substitution at the 4 position with carboxyalkylthio ethers
(26, 28, 29) is also tolerated by the receptor. These target
structures were designed on the basis of molecular modeling
results to engage an electrostatic interaction with K204(7.36)
(Figure 1f).
Our molecular modeling studies suggested that a pure (S)-
conformation (P ) 180°, 2′-endo and 3′-exo) would confer to
the phosphate and nucleobase moieties of UDP the most
favorable orientation for establishment of interactions with the
residues from TM1, TM3, TM6, and TM7. Compound 6, which
is locked in the (S)-conformation (P ) -162°, 3′-exo) by a
(S)-methanocarba (mc) ring, proved to be about 10 times more
potent than 12. Conversely, 5, locked in the (N)-conformation
by a (N)-methanocarba ring, was found to be completely
inactive. Compound 19, which is locked in a (S)-conformation
(P ) 126°, 1′-exo) by a 2-OBH ring system but is further away
from pure (S)-conformation than 6, maintained agonist activity
at the P2Y6 receptor but was less potent than the corresponding
flexible analogues. The proposed conformation of 19 was
calculated using quenched molecular dynamics and energy
minimization. The cyclopropane ring, fused between C3′ and
C4′, forces the C1′ into the exo conformation in order for the
six-membered ring to adopt a pseudoboat conformation.
Table 2 shows the relative EC50 values with respect to the
native ligands at three P2Y receptors for the same changes in
the base or nucleotide, applied to the 5′-diphosphate derivative
(P2Y6 receptor) and to the 5′-triphosphate derivative (P2Y2 and
P2Y4 receptors). In most cases, there is a parallel reduction of
potency at P2Y2 and P2Y4 receptors. However, there tended to
be a greater loss of potency at the P2Y6 receptor, for example,
for 2′-ribose modifications. The removal of the 2′- or 3′-hydroxyl
group was more poorly tolerated at the P2Y6 receptor than at
the two other subtypes. The reduction of potency of the 2′-
deoxyarabino-2′-fluoro analogue 18 was particularly pronounced
at the P2Y6 receptor. The low potency of the 2′-amino analogue
16 is in sharp contrast with the high potency of the correspond-
ing triphosphate at the P2Y2 receptor.37 The 2- and 4-thio
Discussion
As a result of mutagenesis and molecular modeling studies,
we proposed that nucleotides bind to the P2Y receptor with the
sugar moiety accommodated between TM3 and TM7, with the
base pointing toward TM1 and TM2 and with the polyphosphate
moiety pointing in the direction of TM6 (Figure 1a).11,35,36
According to this model, the electrostatic interaction of the
phosphate moiety with three cationic residues from TM3, TM6,
and TM7 (3.29, 6.55, and 7.39) is fundamental for the
recognition of nucleotides. Our current modeling data (Figure
1) suggest that the â-phosphate of UDP and related analogues
is fundamental for the establishment of electrostatic interactions
with two of the three cationic residues, namely, K259(6.55) and
R287(7.39). Not surprisingly, compounds 11, 14, 17, and 33,
which lack a â-phosphate linked through the 5′ position of
ribose, were inactive at the P2Y6 receptor.
The puckering of the ribose ring, described by the phase angle
of pseudorotation (P, Figure 2), is of fundamental importance
for the biological activity of nucleosides and nucleotides. We
recently discovered that the (S)-conformation of the ribose
moiety is preferred for ligand recognition by the P2Y6 receptor.35

Uridine 5′-Diphosphate Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5537
Figure 1. Molecular models of the complexes of the human P2Y6 receptor with UDP analogues 32 (a and b, 5-iodo), 6 (c, (S)-methanocarba), 31
(d, 3-methyl), 23 (e, 4-ethylthio), and 28 (f, 4-carboxyethylthio). In all cases, the diphosphate moiety of the nucleotide is coordinated by three
cationic residues from TM3 (3.29), TM6 (6.55), and TM7 (7.39). The donation of an H-bond from the NH at position 3 of the uracil ring to
Y33(1.39) seems to contribute to higher potency in the activation of the P2Y6 receptor. To facilitate the comparison, in the six panels of this figure
the receptor maintains the same orientation relative to the plane of the membrane. In the schematic representation of the P2Y6 receptor complexed
with 32 (a), the tube represents the backbone of the receptor and is colored according to residue positions, with a spectrum of colors that ranges
from red (N-terminus) to purple (C-terminus): TM1 is in orange, TM2 in ochre, TM3 in yellow, TM4 in green, TM5 in cyan, TM6 in blue, TM7
in purple.
modifications tended to preserve potency at all subtypes but
most effectively at the P2Y2 and P2Y4 receptors. Strikingly,
the 5-iodo modification preserves potency most effectively at
the P2Y6 receptor, and 5-halo substitution of the uracil ring may
therefore be a basis for achieving selectivity at this subtype.
Conclusions
The P2Y receptor family is unusual in comparison to other
pharmacologically defined clusters of GPCR sequences because
they respond to varied and diverse nucleotide ligands. Within
the same phylogenetic cluster are receptors for other anionic

5538 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18
Besada et al.
assignment of the signals was performed by COSY experiments
obtained with the Varian XL 300 spectrometer.
The purity of compounds was checked using a Hewlett-Packard
1100 HPLC equipped with a Luna 5 µm RP-C18(2) analytical
column (250 mm × 4.6 mm; Phenomenex, Torrance, CA). System
A parameters were as follows: linear gradient solvent system of 5
mM TBAP (tetrabutylammonium dihydrogenphosphate)-CH3CN
from 80:20 to 40:60 in 20 min, then isocratic for 2 min; the flow
rate was 1 mL/min. System B parameters were as follows: linear
gradient solvent system of 10 mM TEAA (triethylammonium
acetate)-CH3CN from 100:0 to 85:15 in 20 min, then isocratic for
2 min; the flow rate was 1 mL/min. System C parameters were as
follows: linear gradient solvent system of 10 mM TEAA-CH3-
CN from 90:10 to 60:40 in 20 min, then isocratic for 2 min; the
flow rate was 1 mL/min. The purity of compounds 13, 15-17, 20,
25, 29, 32, 50, and 51 in system B or C was checked using a Zorbax
Eclipse 5 µm XDB-C18 analytical column (250 mm × 4.6 mm;
Agilent Technologies Inc., Palo Alto, CA). Peaks were detected
by UV absorption with a diode array detector. All derivatives tested
for biological activity showed >98% purity in the HPLC systems.
High-resolution mass measurements were performed on Micro-
mass/Waters LCT Premier electrospray time-of-flight (TOF) mass
spectometer coupled with a Waters HPLC system. Purification of
the nucleotide analogues for biological testing was carried out on
(diethylamino)ethyl (DEAE) A-25 Sephadex columns as described
below. Compounds 13 and 15-17 were additionally purified by
HPLC using system D (10 mM TEAA-CH3CN from 100:0 to 90:
10 in 30 min, then isocratic for 2 min; the flow rate was 2 mL/
min) with a Luna 5 µm RP-C18(2) semipreparative column (250
mm × 10.0 mm; Phenomenex, Torrance, CA).
Figure 2. Graphical representation of the pseudorotational cycle,48
which defines all of the possible ribose ring conformations. The phase
angle of pseudorotation (P) describes the geometry of the ribose
puckering. The (N)-methanocarba ring system adopts a C2′-exo
conformation, and the (S)-methanocarba ring system adopts a C3′-exo
conformation. The 2-OBH ring system maintains a C1′-exo conforma-
tion, in the (S)-region.
ligands: lysophosphatidylserine (GPR34), succinic acid (GPR91),
and R-keto-glutarate (GPR80).1,11 The challenge for the me-
dicinal chemist is to understand the structural basis for the P2Y
ligand selectivities and to use that information to design much
needed pharmacological probes to act as selective agonists and
antagonists. We have recently focused on the influence of the
ribose conformation on subtype selectivity. In this study of SAR
at the human P2Y6 receptor following diverse structural changes
of UDP, we have identified various modifications that deselect
for this receptor subtype in relation to other uracil nucleotide-
responsive receptors and several modifications (i.e., 5-iodo and
the (S)-mc ring) that provide P2Y6 selectivity. Combinations
of these various modifications should achieve higher potency
and selectivity and in conjunction with molecular modeling and
receptor docking studies should lead to high-affinity agonists
that are highly selective for the P2Y6 receptor.
General Procedure for the Preparation of Nucleoside 5′-
Diphosphates. Procedure A. A solution of the corresponding
nucleoside (0.04-0.19 mmol) and Proton Sponge (1.5 equiv) in
trimethyl phosphate (2 mL) was stirred for 10 min at 0 °C. Then
phosphorus oxychloride (2 equiv) was added dropwise, and the
reaction mixture was stirred for 2 h at 0 °C. A mixture of
tributylamine (9 equiv) and a solution of 0.35 M bis(tributylam-
monium) salt of phosphoric acid in DMF (6 equiv) was added at
once. This salt was prepared by mixing tributylamine (0.16 mL,
0.66 mmol) and phosphoric acid (34 mg, 0.35 mmol) in DMF (1
mL). After 6 min, 0.2 M triethylammonium bicarbonate solution
(3 mL) was added, and the clear solution was stirred at room
temperature for 45 min. The latter was lyophilized overnight. The
residue was purified by ion-exchange column chromatography using
a Sephadex-DEAE A-25 resin with a linear gradient (0.01-0.5 M)
of 0.5 M ammonium bicarbonate as the mobile phase. The
corresponding nucleoside diphosphates were collected, frozen, and
lyophilized as the ammonium salts.
Experimental Section
(2R,3R,5S)-1-(5-(Diphosphoryloxymethyl)-3-hydroxytetrahy-
drofuran-2-yl)pyrimidine-2,4(1H,3H)-dione, Triethylammonium
Salt (13). Procedure A. Compound 13 (2 mg, 7%) was obtained
Chemical Synthesis. 2′-Deoxyuridine-5′-diphosphate (12) was
purchased from Jena Bioscience USA (San Diego, CA). 3′-
Deoxyuridine-5′-triphosphate was purchased from TriLink Bio-
Technologies (San Diego, CA). 3′-Deoxyuridine (38) was purchased
from T.R.C., Inc. (North York, Ontario, Canada). 2′-Azido-2′-
deoxyuridine (39) and 2′-amino-2′-deoxyuridine (40) were pur-
chased from CMS Chemicals Ltd. (Oxfordshire, U.K.). 2′-Arafluoro-
2′-deoxyuridine (42b) was purchased from R.I. Chemical, Inc.
(Orange, CA). 2-Thiouridine (43) was purchased from Berry &
Associates, Inc. (Dexter, MI). Compounds 21, 47, and 53, reagents,
and solvents were purchased from Sigma-Aldrich (St. Louis, MO).
Compounds 5, 6, 14, and 31 were synthesized in our laboratory as
described.23,32,35,37 Compounds 34, 37, and 44 were synthesized as
reported.25,38,39
1H NMR spectra were obtained with a Varian Gemini 300
spectrometer using D2O as a solvent. The chemical shifts are
expressed as relative ppm from HOD (4.78 ppm). 13C NMR spectra
were recorded at room temperature by use of the Varian XL 300
spectrometer (75 MHz); methanol-d4 was used as an external
standard. 31P NMR spectra were recorded at room temperature by
use of the Varian XL 300 spectrometer (121.42 MHz); orthophos-
phoric acid (85%) was used as an external standard. The complete
1
as a white solid from 38 (10 mg, 0.04 mmol). H NMR (D2O) δ
8.04 (d, J ) 8.1 Hz, 1H), 5.94 (d, J ) 8.1 Hz, 1H), 5.85 (d, J )
1.5 Hz, 1H), 4.68 (m, 1H), 4.55 (m, 1H), 4.35 (m, 1H), 4.14 (m,
1H), 2.25 (m, 1H), 2.10 (m, 1H); 31P NMR (D2O) δ -10.33 (d, J
) 20.8 Hz), -10.79 (d, J ) 19.6 Hz); HRMS m/z found 386.9995
(M - H+)-. C9H13N2O11P2 requires 386.9995; HPLC (system A)
13.7 min (98%), (system B) 6.7 min (98%).
(2R,3R,4S,5R)-1-(3-Azido-5-(diphosphoryloxymethyl)-4-hy-
droxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione, Tri-
ethylammonium Salt (15). Procedure A. Compound 15 (12 mg,
35%) was obtained as a white solid from 39 (32 mg, 0.12 mmol).
1H NMR (D2O) δ 7.99 (d, J ) 8.1 Hz, 1H), 6.04 (d, J ) 5.1 Hz,
1H), 5.99 (d, J ) 8.1 Hz, 1H), 4.63 (m, 1H), 4.40 (m, 1H), 4.29
(m, 1H), 4.25 (m, 2H); 31P NMR (D2O) δ -9.63, -10.81; HRMS
m/z found 427.9993 (M - H+)-. C9H12N5O11P2 requires 428.0009;
HPLC (system A) 13.9 min (99%), (system B) 9.8 min (99%).
(2R,3R,4S,5R)-N-(2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)-2,2,2-tri-
fluoroacetamide (41). To a solution of 40 (20 mg, 0.08 mmol) in
DMF (1 mL) was added DIEA (0.04 mL, 0.25 mmol) and ethyl

Uridine 5′-Diphosphate Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5539
Table 2. Relative Effects of Parallel Structural Modifications of Uracil Nucleotide Analogues on Their Potency in the Stimulation of PLC at
Recombinant Human P2Y2, P2Y4, and P2Y6 Receptorsa
EC50 ratio of
5′-diphosphate/UDP,
hP2Y6 receptor
EC50 ratio of
5′-triphosphate/UTP,
hP2Y2 receptor
EC50 ratio of
5′-triphosphate/UTP,
hP2Y4 receptor
compd no. of
5′-diphosphate
modification
(as di- or triphosphate)
structuree
substitution of base moiety
1
7
8
9
UDP, UTP
e
e
e
e
e
1
1
1
CDP, CTP
ADP, ATP
GDP, GTP
IDP, ITP
1000b
>7000
500b
400b
110
1.7
54
NAg
NAg
90
10
13c
9.1c
ribose modifications
5
12
13
15
16
18
(N)-methanocarba
2′-deoxy
3′-deoxy
2′-azido-2′-deoxy
2′-amino-2′-deoxy
2′-deoxyarabino-2′-fluoro-
e
>7000
130
190
120
300
2.0d
22
1.7d
26
R7 ) H
R8 ) H
17f
100
1.3
11
42f
15
R7 ) N3
R7 ) NH2
R7 ) H, R6 ) F
16
7.1
420
uracil modifications
20
21
31
32h
2-thio
4-thio
3-methyl
5-iodo
R2 ) S
R4 ) S
R3 ) CH3
R5 ) I
4
6
250
1.1
0.71
0.53
24
4.8
0.32
47
17
55
a The EC50 ratios at the P2Y6 receptors are derived from the data in Table 1 for derivatives of the native agonist, i.e., 5′-diphosphates. The EC50 ratios
of the corresponding 5′-triphosphates at human P2Y2 and P2Y4 receptors expressed in astrocytoma cells are derived from published data,23,37 unless otherwise
noted. In each case, the endogenous agonist (UDP at P2Y6 receptors, UTP at P2Y2 and P2Y4 receptors) is assigned the value of 1. b Robaye et al., 1997.24
c Kennedy et al., 2000.47 d Kim et al., 2002.23 e See Table 1 and Chart 1. f The EC50 values of 3′-deoxy-UTP were determined to be 0.81 ( 0.09 µM at
P2Y2 receptors and 3.1 ( 1.1 µM at P2Y4 receptors, using reported methods.37 g NA: no activation at 10 µM. h 32, MRS2693.
trifluoroacetate (0.03 mL, 0.25 mmol). The reaction mixture was
stirred at room temperature for 16 h. The solvent was removed in
vacuo, and the residue was purified by preparative thin-layer
chromatography (CH2Cl2-MeOH, 85:15) to afford 41 (24 mg,
found 404.9892 (M - H+)-. C9H12N2O11P2F requires 404.9900;
HPLC (system A) 15.1 min (99%), (system B) 10.9 min (99%).
(1S,3R,4R,5S)-1-(4-Hydroxy-1-(diphosphoryloxymethyl)-2-
oxabicyclo[3.1.0]hexan-3-yl)pyrimidine-2,4(1H,3H)-dione, Am-
monium Salt (19). Procedure A. Compound 19 (4 mg, 31%) was
1
86%) as a white solid. H NMR (CD3OD) δ 8.01 (d, J ) 8.1 Hz,
1
1H), 6.13 (d, J ) 7.8 Hz, 1H), 5.73 (d, J ) 8.1 Hz, 1H), 4.60 (m,
1H), 4.31 (m, 1H), 4.01 (m, 1H), 3.77 (m, 2H); HRMS m/z found
338.0587 (M - H+)-. C11H11N3O6F3 requires 338.0600.
obtained as a white solid from 44 (10 mg, 0.04 mmol). H NMR
(D2O) δ 7.80 (d, J ) 8.1 Hz, 1H), 5.79 (d, J ) 8.1 Hz, 1H), 5.60
(d, J ) 8.1 Hz, 1H), 4.65 (m, 1H), 4.07(m, 1H), 3.67 (m, 1H),
1.81 (m, 1H), 1.37 (m, 1H), 0.84 (m, 1H); 31P NMR (D2O) δ -7.08,
-10.55; HRMS m/z found 398.9985 (M - H+)-. C10H13N2O11P2
requires 398.9995; HPLC (system A) 14.5 min (99%), (system B)
10.2 min (99%).
(2R,3R,4S,5R)-1-(3,4-Dihydroxy-5-(diphosphoryloxymethyl)-
tetrahydrofuran-2-yl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-
one, Ammonium Salt (20). Procedure A. Compound 20 (21 mg,
23%) was obtained as a white solid from 38 (50 mg, 0.19 mmol).
1H NMR (D2O) δ 8.19 (d, J ) 8.1 Hz, 1H), 6.65 (d, J ) 2.7 Hz,
1H), 6.27 (d, J ) 8.1 Hz, 1H), 4.45 (m, 1H), 4.32 (m, 4H); 31P
NMR (D2O) δ -9.35 (d, J ) 20.8 Hz), -10.84 (d, J ) 20.8 Hz);
HRMS m/z found 418.9712 (M - H+)-. C9H13N2O11P2S requires
418.9715; HPLC (system A) 15.0 min (98%), (system B) 8.4 min
(98%).
General Procedure for the Preparation of 4-Substituted-thio-
UDP Analogues. Procedure B. To a suspension of 4-thiouridine
5′-diphosphate (5 mg, 0.01 mmol) in MeOH (1 mL) was added
0.25 M NaOH (0.14 mL). The reaction mixture was stirred at room
temperature for 2 h, and then the solvent was eliminated under
high vacuum. The 4-thio-UDP sodium salt obtained was suspended
in dry DMF (1.5 mL), and an excess of the corresponding alkyl
halide (50 equiv) was added. The reaction mixture was stirred at
room temperature for 24 h, and the progress of the reaction was
monitored by analytical HPLC (system A). After removal of the
solvent, the residue was purified using the same method as in
procedure A, and the corresponding nucleotide diphosphates were
obtained as the ammonium salts.
(2R,3R,4S,5R)-1-(3-Amino-5-(diphosphoryloxymethyl)-4-hy-
droxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (16).
Procedure A. Compound 16 in the triethylammonium salt (7 mg,
17%) was obtained as a white solid from 41 (20 mg, 0.06 mmol).
Alternatively, compound 16 (0.6 mg, 75%) was obtained by
treatment of a solution of the triethylammonium salt of compound
15 (1.5 mg, 0.002 mmol) in MeOH (1 mL) with 10% Pd/C (0.5
mg) and H2 at atmospheric pressure for 1 h at room temperature.
1H NMR (D2O) δ 7.99 (d, J ) 7.5 Hz, 1H, H-6 pyrimidine), 6.12
(d, J ) 7.2 Hz, 1H, H-2), 6.00 (d, J ) 7.5 Hz, 1H, H-5 pyrimidine),
4.61 (m, 1H, H-4), 4.36 (m, 1H, H-5), 4.24 (m, 1H, HCHO), 4.16
(m, 1H, HCHO), 3.88 (m, 1H, H-3); 31P NMR (D2O) δ -6.77,
-10.57; HRMS m/z found 402.0126 (M - H+)-. C9H14N3O11P2
requires 402.0104; HPLC (system A) 6.0 min (99%), (system B)
7.1 min (99%).
(2R,3R,4S,5R)-1-(3-Amino-4-(diphosphoryloxy)-5-(hydroxy-
methyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione, Tri-
ethylammonium Salt (17). Procedure A. Compound 17 (6 mg,
8%) was obtained as a white solid from 40 (25 mg, 0.10 mmol).
1H NMR (D2O) δ 7.88 (d, J ) 8.1 Hz, 1H, H-6 pyrimidine), 6.29
(d, J ) 6.9 Hz, 1H, H-2), 5.91 (d, J ) 8.1 Hz, 1H, H-5 pyrimidine),
5.01 (m, 1H, H-4), 4.46 (m, 1H, H-5), 4.14 (m, 1H, H-3), 3.89 (m,
2H, CH2O); 13C NMR (D2O) δ 167.3, 152.9, 142.4, 103.9, 87.9,
87.5, 74.7, 62.0, 56.3; 31P NMR (D2O) δ -7.73 (d, J ) 22.0 Hz),
-11.16 (d, J ) 22.0 Hz); HRMS m/z found 402.0094 (M - H+)-.
C9H14N3O11P2 requires 402.0104; HPLC (system A) 10.6 min
(98%), (system B) 6.7 min (98%).
(2R,3S,4R,5R)-1-(5-(Diphosphoryloxymethyl)-3-fluoro-4-hy-
droxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione, Am-
monium Salt (18). Procedure A. Compound 18 (2.1 mg, 32%)
Procedure C. To a solution of 4-thiouridine 5′-monophosphate
(10 mg, 0.03 mmol) in MeOH (1 mL) was added 0.25 M NaOH
(0.13 mL). The reaction mixture was stirred at room temperature
for 2 h, and then the solvent was eliminated under high vacuum.
The 4-thio-UMP sodium salt obtained was suspended in dry DMF
(2 mL), and an excess of the corresponding alkyl halide (50 equiv)
was added. The reaction mixture was stirred at 90 °C for 4-8 h,
1
was obtained as a white solid from 42b (9 mg, 0.04 mmol). H
NMR (D2O) δ 7.79 (d, J ) 7.9 Hz, 1H), 6.36 (m, 1H), 5.93 (d, J
) 7.9 Hz, 1H), 5.53 (apparent dt, JH-F ) 69.0 Hz, 1H), 4.42 (m,
1H), 4.19 (m, 3H); 31P NMR (D2O) δ -8.89, -10.81; HRMS m/z

5540 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18
Besada et al.
and the progress of the reaction was monitored by analytical HPLC
(system A). The solvent was removed in vacuo to obtain the
corresponding 4-substituted-thio-UMP as a crude solid, which then
was used directly in the next step without further purification. To
a solution of the crude solid containing 4-substituted-thio-UMP in
DMF (1.5 mL) was added 1,1′-carbonyldiimidazole (26 mg, 0.16
mmol). The reaction mixture was stirred at room temperature for
6 h. Then 5% triethylamine solution in 1/1 water/methanol (3 mL)
was added and stirring was continued at room temperature for an
additional 2 h. After removal of the solvent, the residue was dried
in high vacuum and dissolved in DMF (1 mL). To this solution
was successively added tributylamine (0.11 mL, 0.46 mmol) and a
solution of 0.35 M bis(tributylammonium) salt of phosphoric acid
in DMF (0.4 mL). The reaction mixture was stirred at room
temperature for 2 days, and then 0.2 M triethylammonium
bicarbonate was added. After removal of the solvent, the residue
was purified using the same method as in procedure A, and the
corresponding nucleotide diphosphates were obtained as the am-
monium salts.
7.1 Hz, 1H), 6.75 (d, J ) 7.1 Hz, 1H), 5.96 (d, J ) 2.4 Hz, 1H),
4.35 (m, 4H), 4.25 (m, 1H), 3.88 (s, 2H); 31P NMR (D2O) δ -10.37
(d, J ) 20.6 Hz), -10.92 (d, J ) 20.6 Hz); HRMS m/z found
476.9773 (M - H+)-. C11H15N2O13P2S requires 476.9770; HPLC
(system A) 18.3 min (99%), (system B) 9.0 min (99%).
(2R,3R,4S,5R)-2-(1-(3,4-Dihydroxy-5-(diphosphoryloxymeth-
yl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylthio)-
acetamide, Ammonium Salt (27). Procedure B. Compound 27
(3.8 mg, 79%) was obtained as a white solid using iodoacetamide
1
as alkylating agent. H NMR (D2O) δ 8.31 (d, J ) 7.4 Hz, 1H),
6.79 (d, J ) 7.4 Hz, 1H), 5.92 (d, J ) 1.5 Hz, 1H), 4.42 (m, 1H),
4.32 (m, 4H), 3.96 (s, 2H); 13C NMR (D2O) δ 177.9, 173.9, 156.0,
141.9, 105.8, 91.2, 83.0, 75.1, 68.3, 63.8, 33.0; 31P NMR (D2O) δ
-7.63, -10.66 (d, J ) 21.9 Hz); HRMS m/z found 475.9935 (M
- H+)-. C11H16N3O12P2S requires 475.9930; HPLC (system A) 14.6
min (99%), (system B) 8.6 min (99%).
(2R,3R,4S,5R)-3-(1-(3,4-Dihydroxy-5-(diphosphoryloxymeth-
yl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylthio)-
propanoic Acid, Ammonium Salt (28). Procedure C. Compound
49 (HPLC (system A) 16.0 min) was obtained as a crude solid
using methyl 3-bromopropionate and stirring at 90 °C for 8 h in
the alkylation reaction. The residue that contained compound 49
was dissolved in H2O (1 mL), and 0.25 M NaOH (2 mL) was added.
The reaction mixture was stirred at room temperature for 6 h. After
removal of the solvent, the residue was purified following the
general procedure to give compound 28 (2.8 mg, 20% from 47) as
a white solid. 1H NMR (D2O) δ 8.21 (d, J ) 7.2 Hz, 1H), 6.76 (d,
J ) 7.2 Hz, 1H), 5.99 (d, J ) 2.7 Hz, 1H), 4.38 (m, 4H), 4.26 (m,
1H), 3.39 (t, J ) 6.9 Hz, 2H), 2.73 (t, J ) 6.9 Hz, 2H); 31P NMR
(D2O) δ -10.39, -10.84; HRMS m/z found 490.9951 (M - H+)-.
C12H17N2O13P2S requires 490.9927; HPLC (system A) 18.5 min
(99%), (system B) 9.6 min (99%).
(2R,3R,4S,5R)-4-(1-(3,4-Dihydroxy-5-(diphosphoryloxymeth-
yl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylthio)-
butanoic Acid, Ammonium Salt (29). Procedure B. Compound
46 (HPLC (system A) 16.4 min) was obtained as a crude solid
using methyl 4-iodobutyrate as alkylating agent and stirring the
reaction mixture at 50 °C for 20 h. The residue that contained
compound 46 was dissolved in H2O (2 mL), and 0.25 M NaOH (2
mL) was added. The reaction mixture was stirred at room
temperature for 2 h. After removal of the solvent, the residue was
purified following the general procedure to give compound 29 (2.0
mg, 16% from 21) as a white solid. 1H NMR (D2O) δ 8.19 (d, J )
6.8 Hz, 1H), 6.75 (d, J ) 6.8 Hz, 1H), 5.96 (m, 1H), 4.35 (m, 4H),
4.24 (m, 1H), 3.19 (d, J ) 7.1 Hz, 1H), 2.40 (d, J ) 7.5 Hz, 1H),
1.99 (m, 2H); 31P NMR (D2O) δ -10.22, -10.98; HRMS m/z found
505.0096 (M - H+)-. C13H19N2O13P2S requires 505.0083; HPLC
(system A) 18.2 min (99%), (system B) 9.2 min (99%).
(2R,3R,4S,5R)-1-(3,4-Dihydroxy-5-(monophosphoryloxymeth-
yl)tetrahydrofuran-2-yl)-4-(hexylthio)pyrimidin-2(1H)-one, Am-
monium Salt (50). To a solution of 4-thiouridine 5′-monophosphate
(10 mg, 0.03 mmol) in MeOH (1 mL) was added 0.25 M NaOH
(0.13 mL). The reaction mixture was stirred at room temperature
for 2 h, and then the solvent was eliminated under high vacuum.
The 4-thio-UMP sodium salt obtained was suspended in dry DMF
(2 mL), and 1-iodohexane (0.19 mL, 1.28 mmol) was added. The
reaction mixture was stirred at 90 °C for 4 h, and the progress of
the reaction was monitored by analytical HPLC (system A). The
solvent was removed in vacuo to obtain compound 50 as a crude
solid, which then was used directly in the next step without further
purification. For characterization purposes a small portion of
compound 50 was purified by ion-exchange column chromatog-
raphy as described in procedure A. 1H NMR (D2O) δ 8.21 (d, J )
7.5 Hz, 1H), 6.73 (d, J ) 7.5 Hz, 1H), 5.97 (m, 1H), 4.34 (m, 3H),
4.29 (m, 1H), 4.14 (m, 1H), 3.18 (t, J ) 7.1 Hz, 2H), 1.74 (m,
2H), 1.46 (m, 2H), 1.33 (m, 4H), 0.89 (t, J ) 6.9 Hz, 3H); 31P
NMR (D2O) δ 0.60; HRMS m/z found 423.0995 (M - H+)-.
C15H24N2O8PS requires 423.0991; HPLC (system A) 15.1 min
(99%), (system C) 13.7 min (99%).
(2R,3R,4S,5R)-1-(3,4-Dihydroxy-5-(diphosphoryloxymethyl)-
tetrahydrofuran-2-yl)-4-(methylthio)pyrimidin-2(1H)-one, Am-
monium Salt (22). Procedure B. Compound 22 (7 mg, 80%) was
obtained as a white solid from 21 (10 mg, 0.02 mmol) and using
1
iodomethane as alkylating agent. H NMR (D2O) δ 8.25 (d, J )
7.1 Hz, 1H), 6.76 (d, J ) 7.1 Hz, 1H), 5.94 (d, J ) 2.4 Hz, 1H),
4.41 (m, 1H), 4.32 (m, 4H), 2.54 (s, 3H); 13C NMR (D2O) δ 182.4,
156.8, 142.0, 106.4, 91.9, 83.8, 76.0, 69.6, 65.0, 14.0; 31P NMR
(D2O) δ -7.15 (d, J ) 23.1 Hz), -10.67 (d, J ) 23.1 Hz); HRMS
m/z found 432.9888 (M - H+)-. C10H15N2O11P2S requires 432.9872;
HPLC (system A) 14.9 min (99%), (system B) 11.1 min (99%).
(2R,3R,4S,5R)-1-(3,4-Dihydroxy-5-(diphosphoryloxymethyl)-
tetrahydrofuran-2-yl)-4-(ethylthio)pyrimidin-2(1H)-one, Am-
monium Salt (23). Procedure B. Compound 23 (2 mg, 44%) was
obtained as a white solid using iodoethane as alkylating agent and
1
stirring the reaction mixture at 70 °C for 4 h. H NMR (D2O) δ
8.24 (d, J ) 7.4 Hz, 1H), 6.75 (d, J ) 7.4 Hz, 1H), 5.94 (d, J )
2.4 Hz, 1H), 4.41 (m, 1H), 4.32 (m, 4H), 3.15 (q, J ) 7.5 Hz, 2H),
1.36 (t, J ) 7.5 Hz, 3H); 31P NMR (D2O) δ -7.49, -10.73; HRMS
m/z found 447.0032 (M - H+)-. C11H17N2O11P2S requires 447.0028;
HPLC (system A) 14.7 min (99%), (system B) 14.5 min (99%).
(2R,3R,4S,5R)-4-(Allylthio)-1-(3,4-dihydroxy-5-(diphosphory-
loxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one, Ammo-
nium Salt (24). Procedure B. Compound 24 (3.4 mg, 73%) was
obtained as a white solid using allyl bromide as alkylating agent.
1H NMR (D2O) δ 8.26 (d, J ) 7.5 Hz, 1H), 6.76 (d, J ) 7.5 Hz,
1H), 5.99 (m, 1H), 5.93 (m, 1H), 5.40 (d, J ) 17.1 Hz, 2H), 5.23
(d, J ) 10.2 Hz, 2H), 4.43 (m, 1H), 4.32 (m, 4H), 3.84 (d, J ) 6.6
Hz, 2H); 31P NMR (D2O) δ -6.08 (d, J ) 21.9 Hz), -10.55 (d, J
) 21.9 Hz); HRMS m/z found 459.0039 (M - H+)-. C12H17-
N2O11P2S requires 459.0028; HPLC (system A) 16.0 min (99%),
(system B) 16.2 min (99%).
(2R,3R,4S,5R)-4-(Benzylthio)-1-(3,4-dihydroxy-5-(diphospho-
ryloxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one, Am-
monium Salt (25). Procedure B. Compound 25 (3.3 mg, 65%)
was obtained as a white solid using benzyl bromide as alkylating
1
agent. H NMR (D2O) δ 8.21 (d, J ) 7.2 Hz, 1H), 7.52 (m, 2H),
7.39 (m, 3H), 6.73 (d, J ) 7.2 Hz, 1H), 5.95 (d, J ) 2.7 Hz, 1H),
4.46 (s, 2H), 4.34 (m, 4H), 4.23 (m, 1H); 31P NMR (D2O) δ -9.41,
-10.93; HRMS m/z found 509.0171 (M - H+)-. C16H19N2O11P2S
requires 509.0185; HPLC (system A) 16.8 min (98%), (system C)
7.9 min (98%).
(2R,3R,4S,5R)-2-(1-(3,4-Dihydroxy-5-(diphosphoryloxymeth-
yl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylthio)-
acetic Acid, Ammonium Salt (26). Procedure B. Compound 45
(HPLC (system A) 15.6 min) was obtained as a crude solid using
methyl bromoacetate as alkylating agent. The residue that contained
compound 45 was dissolved in H2O (1 mL), and 0.25 M NaOH
(0.5 mL) was added. The reaction mixture was stirred at room
temperature for 2 h. After removal of the solvent, the residue was
purified following the general procedure to give compound 26 (2.4
mg, 49% from 21) as a white solid. 1H NMR (D2O) δ 8.21 (d, J )
(2R,3R,4S,5R)-1-(3,4-Dihydroxy-5-(diphosphoryloxymethyl)-
tetrahydrofuran-2-yl)-4-(hexylthio)pyrimidin-2(1H)-one, Am-

Uridine 5′-Diphosphate Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5541
monium Salt (30). To a solution of the crude solid containing
compound 50 in DMF (1.5 mL) was added 1,1′-carbonyldiimidazole
(26 mg, 0.16 mmol). The reaction mixture was stirred at room
temperature for 6 h. Then 5% triethylamine solution in 1/1 water/
methanol (3 mL) was added and stirring was continued at room
temperature for an additional 2 h. After removal of the solvent,
the residue was dried in high vacuum and dissolved in DMF (1
mL). To this solution was successively added tributylamine (0.11
mL, 0.46 mmol) and a solution of 0.35 M bis(tributylammonium)
salt of phosphoric acid in DMF (0.4 mL). The reaction mixture
was stirred at room temperature for 2 days, and then 0.2 M
triethylammonium bicarbonate was added. After removal of the
solvent, the residue was purified using the same method as in
procedure A to afford 30 (3.4 mg, 21% from 47) as a white solid.
1H NMR (D2O) δ 8.21 (d, J ) 7.4 Hz, 1H), 6.75 (d, J ) 7.4 Hz,
1H), 5.96 (d, J ) 2.7 Hz, 1H), 4.36 (m, 4H), 4.25 (m, 1H), 3.17
(t, J ) 7.2 Hz, 2H), 1.73 (m, 2H), 1.45 (m, 2H), 1.32 (m, 4H),
0.88 (t, J ) 7.1 Hz, 3H); 31P NMR (D2O) δ -9.67, -10.83;
HRMS m/z found 503.0606 (M - H+)-. C15H25N2O11P2S requires
503.0654; HPLC (system A) 18.9 min (99%), (system C) 13.8 min
(99%).
temperature for 3 d, and then 0.2 M triethylammonium bicarbonate
(2 mL) was added. The mixture was stirred for 30 min. After
removal of the solvent, under reduced pressure at 50 °C, the residue
was purified using the same method as in procedure A to afford
33 (1.6 mg, 29%) as a white solid. 1H NMR (D2O) δ 7.78 (d, J )
8.1 Hz, 1H), 6.35 (dd, J ) 13.1, 5.3 Hz, 1H), 5.92 (d, J ) 8.1 Hz,
1H), 5.50 (apparent dt, JH-F ) 52.2 Hz, 1H), 4.47 (m, 1H), 4.25
(m, 3H); 31P NMR (D2O) δ -7.15, -10.37; HRMS m/z found
386.9783 (M - H+)-. C9H10N2O10P2F requires 386.9795; HPLC
(system A) 13.2 min (99%), (system B) 8.6 min (99%).
(2R,3R,4S,5R)-1-(3,4-Dihydroxy-5-(monophosphoryloxymeth-
yl)tetrahydrofuran-2-yl)-4-(ethylthio)pyrimidin-2(1H)-one, Am-
monium Salt (51). To a solution of 4-thiouridine 5′-monophosphate
(10 mg, 0.03 mmol) in MeOH (1 mL) was added 0.25 M NaOH
(0.13 mL). The reaction mixture was stirred at room temperature
for 2 h, and then the solvent was eliminated under high vacuum.
The 4-thio-UMP sodium salt obtained was suspended in dry DMF
(2 mL), and iodoethane (0.1 mL, 1.28 mmol) was added. The
reaction mixture was stirred at 70 °C for 2 h, and the progress of
the reaction was monitored by analytical HPLC (system A). The
solvent was removed in vacuo to obtain compound 51 as a crude
solid, which then was used directly in the next step without further
purification. For characterization purposes a small portion of
compound 51 was purified by ion-exchange column chromatog-
raphy as described in procedure A. 1H NMR (D2O) δ 8.26 (d, J )
7.4 Hz, 1H), 6.73 (d, J ) 7.4 Hz, 1H), 5.96 (m, 1H), 4.33 (m, 3H),
4.24 (m, 1H), 4.09 (m, 1H), 3.15 (q, J ) 7.4 Hz, 2H), 1.36 (t, J )
7.4 Hz, 3H); 31P NMR (D2O) δ 1.48; HRMS m/z found 367.0377
(M - H+)-. C11H16N2O8PS requires 367.0365; HPLC (system A)
8.5 min (98%), (system B) 6.7 min (98%).
(2R,3R,4S,5R)-1-(3,4-Dihydroxy-5-(diphosphoryloxymethyl)-
tetrahydrofuran-2-yl)-5-iodopyrimidine-2,4(1H,3H)-dione, Am-
monium Salt (32). To a solution of 53 (8 mg, 0.02 mmol) in DMF
(2 mL) was added 1,1′-carbonyldiimidazole (16 mg, 0.10 mmol).
The reaction mixture was stirred at room temperature for 6 h. Then
5% triethylamine solution in 1/1 water/methanol (2 mL) was added
and stirring was continued at room temperature for an additional 2
h. After removal of the solvent, the residue was dried in high
vacuum and dissolved in DMF (2 mL). To this solution was
successively added tributylamine (0.07 mL, 0.29 mmol) and a
solution of 0.35 M bis(tributylammonium) salt of phosphoric acid
in DMF (0.3 mL). The reaction mixture was stirred at room
temperature for 3 days, and then 0.2 M triethylammonium
bicarbonate (2 mL) was added. The mixture was stirred for 30 min.
After removal of the solvent, the residue was purified using the
same method as in procedure A to afford 32 (1.8 mg, 19%) as a
P1,P2-Bis-5-[(2R,3R,4S,5R)-1-(3,4-dihydroxy-5-(hydroxymeth-
yl)tetrahydrofuran-2-yl)-4-(ethylthio)pyrimidin-2(1H)-one]py-
rophosphate, Ammonium Salt, (4-SEt)Up2(4-SEt)U (35). To a
solution of the crude solid containing compound 51 in DMF (1.5
mL) was added 1,1′-carbonyldiimidazole (26 mg, 0.16 mmol). The
reaction mixture was stirred at room temperature for 6 h. Then 5%
triethylamine solution in 1/1 water/methanol (3 mL) was added and
stirring was continued at room temperature for an additional 2 h.
After removal of the solvent, the residue was dried in high vacuum
and dissolved in DMF (1 mL). To this solution was successively
added tributylamine (0.11 mL, 0.46 mmol) and a solution of 0.35
M bis(tributylammonium) salt of phosphoric acid in DMF (0.4 mL).
The reaction mixture was stirred at room temperature for 2 days,
and then 0.2 M triethylammonium bicarbonate was added. After
removal of the solvent, the residue was purified using the same
method as in procedure A to afford 35 (5 mg, 52% from 47) as a
1
white solid. H NMR (D2O) δ 8.28 (s, 1H), 5.93 (d, J ) 4.2 Hz,
1H), 4.42 (m, 2H), 4.25 (m, 3H); 31P NMR (D2O) δ -6.89, -10.60;
HRMS m/z found 528.8901 (M - H+)-. C9H12N2O12P2I requires
528.8910; HPLC (system A) 14.7 min (99%), (system B) 8.8 min
(99%).
(2R,3S,4R,5R)-1-(3-Fluoro-4-hydroxy-5-(monophosphoryloxy-
methyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione, Am-
monium Salt (54). A solution of 2′-fluoro-2′-deoxy-â-D-arabino-
furanosyluridine (15 mg, 0.06 mmol) and Proton Sponge (26 mg,
0.12 mmol) in trimethyl phosphate (2 mL) was stirred for 10 min
at 0 °C. Then phosphorus oxychloride was added dropwise (0.01
mL, 0.12 mmol), and the reaction mixture was stirred for 2 h at 0
°C. Then 0.2 M triethylammonium bicarbonate solution (7 mL)
was added, and the clear solution was stirred at room temperature
for 45 min. The latter was lyophilized overnight. The residue was
purified using the same method as in procedure A to afford 54 (7
mg, 60%) as a white solid. 1H NMR (D2O) δ 7.90 (d, J ) 8.1 Hz,
1H), 6.31 (dd, J ) 15.9, 4.2 Hz, 1H), 5.89 (d, J ) 8.1 Hz, 1H),
1
white solid. H NMR (D2O) δ 8.10 (d, J ) 7.4 Hz, 2H), 6.57 (d,
J ) 7.4 Hz, 2H), 5.95 (d, J ) 2.7 Hz, 2H), 4.45 (m, 2H), 4.28 (m,
8H), 3.10 (q, J ) 7.5 Hz, 4H), 1.35 (t, J ) 7.5 Hz, 6H); 31P NMR
(D2O) δ -10.23; HRMS m/z found 717.0711 (M - H+)-.
C22H31N4O15P2S2 requires 717.0703; HPLC (system A) 14.9 min
(99%), (system C) 6.8 min (99%).
P1,P2-Bis-5-[(2R,3R,4S,5R)-4-(benzylthio)-1-(3,4-dihydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one]py-
rophosphate, Ammonium Salt, (4-SBn)Up2(4-SBn)U (36). Pro-
cedure C. Compound 36 (5 mg, 45% from 47) was obtained as a
white solid using benzyl bromide and stirring overnight at room
5.21 (apparent dt, JH-F ) 51.6 Hz, 1H), 4.52 (apparent dt, JH-F
)
19.2 Hz, 1H), 4.13 (m, 3H); 31P NMR (D2O) δ 4.01; HRMS m/z
found 325.0240 (M - H+)-. C9H11N2O8FP requires 325.0237;
HPLC (system A) 10.1 min (99%), (system B) 6.2 min (99%).
(2R,3S,4R,5R)-1-(3-Fluoro-4-hydroxy-5-(hydroxymethyl)tet-
rahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione Cyclic-4,5-
diphosphate, Ammonium Salt (33). To a solution of 54 (5 mg,
0.02 mmol) in DMF (2 mL) was added 1,1′-carbonyldiimidazole
(12 mg, 0.08 mmol). The reaction mixture was stirred at room
temperature for 8 h. Then 5% triethylamine in methanol was added
and stirring was continued at room temperature for an additional 2
h. After removal of the solvent, the residue was dried in high
vacuum and dissolved in DMF (1 mL). To this solution was
successively added tributylamine (0.06 mL, 0.25 mmol) and a
solution of 0.35 M bis(tributylammonium) salt of phosphoric acid
in DMF (0.23 mL). The reaction mixture was stirred at room
1
temperature in the alkylation reaction. H NMR (D2O) δ 8.11 (d,
J ) 7.2 Hz, 2H), 7.47 (m, 4H), 7.37 (m, 6H), 6.55 (d, J ) 7.2 Hz,
2H), 5.92 (d, J ) 2.4 Hz, 2H), 4.31 (m, 14H); 31P NMR (D2O) δ
-10.81; HRMS m/z found 841.0991 (M - H+)-. C32H35N4O15P2S2
requires 841.1016; HPLC (system A) 19.2 min (99%), (system C)
16.8 min (99%).
Assay of P2Y6 Receptor-Stimulated PLC Activity. A stable
cell line expressing the human P2Y6 receptor in 1321N1 human
astrocytoma cells was generated as previously described in detail.16
Agonist-induced [3H]inositol phosphate production was measured
in 1321N1 cells grown to confluence on 48-well plates. Twelve
hours before the assay, the inositol lipid pool of the cells was
radiolabeled by incubation in 200 µL of serum-free inositol-free
Dulbecco’s modified Eagle’s medium, containing 0.4 µCi of myo-

5542 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18
Besada et al.
[3H]inositol. No changes of medium were made after the addition
of [3H]inositol. On the day of the assay, cells were challenged with
50 µL of the 5-fold concentrated solution of receptor agonists in
200 mM Hepes (N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic
acid), pH 7.3, containing 50 mM LiCl for 20 min at 37 °C.
Incubations were terminated by aspiration of the drug-containing
medium and addition of 450 µL of ice-cold 50 mM formic acid.
After 15 min at 4 °C, samples were neutralized with 150 µL of
150 mM NH4OH. [3H]Inositol phosphates were isolated by ion
exchange chromatography on Dowex AG 1-X8 columns as previ-
ously described.31
Data Analysis. Agonist potencies (EC50 values) were obtained
from concentration response curves by nonlinear regression analysis
using the GraphPad software package Prism (GraphPad, San Diego,
CA). All experiments were performed in triplicate assays and
repeated at least three times. The results are presented as the mean
( SEM of multiple experiments or in the case of concentration
effect curves from a single experiment carried out with triplicate
assays that were representative of results from multiple experiments.
Molecular Modeling. Molecular mechanics calculations have
been carried out by means of the Discover3 module of InsightII,40
using the CFF91 force field.41 The receptor model used for the
docking experiments was the P2Y6-UDP complex that we previ-
ously built11 and optimized by means of a molecular dynamics (MD)
simulation in an explicit fully hydrated lipid bilayer.35
Before the docking experiments were performed, the ligands were
first fully optimized by means of quenched molecular dynamics
followed by energy minimization. An NVT (constant-volume/
constant-temperature) molecular dynamics simulation was carried
out at a constant temperature of 300 K for 10 ps, using a time step
of 1 fs. During the simulation, snapshots of the system were taken
at regular intervals of 1 ps. The structures in each snapshot were
energy-minimized (BFGS Newton method) until a root-mean-
squared deviation (rmsd) of 0.000 01 kcal mol-1 Å-1 on the gradient
was reached. For each ligand, the conformation showing the lowest
energy was promoted to the next phase of the study.
Subsequently, the ligands were flexibly superimposed to the
bound conformation of 1, as derived from our MD simulation.35
The flexible superimpositions have been carried out by means of
the FieldFit program of the Search Compare module of InsightII,40
giving equal weight to the steric and the electrostatic factors. The
bound conformation of 1 was kept rigid during the calculation, while
the structures to be superimposed were fully flexible. An alignment
of the molecules based on their dipole and quadruple moments was
used as the starting position.
financial support. Mass spectral measurements were carried out
by Dr. John Lloyd and NMR by Wesley White (NIDDK). We
thank Kanna Palaniappan for proofreading this manuscript. This
research was supported in part by the Intramural Research
Program of the NIH, National Institute of Diabetes and Digestive
and Kidney Diseases. This work was supported by National
Institutes of Health Grants GM38213 and HL34322 to T. K.
Harden.
Supporting Information Available: Coordinates of the com-
plex of the human P2Y6 receptor with compound 32 (5-iodoUDP)
in pdb format. This material is available free of charge via the
Internet at http://pubs.acs.org.
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The docking experiments were carried out by means of the Monte
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of InsightII.40 The binding site was defined as all the residues within
a distance of 6 Å from the ligand. Full flexibility was granted to
the ligands and to the residues of the binding site. The scaling factor
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term was set at 1. The maximum movement in each random
translation and rotation of the ligand were set to 0.1 Å and 1°,
respectively.
After the docking procedure, the receptor-ligand complexes
were optimized by means of a Monte Carlo multiple minimum
(MCMM)42 conformational search as implemented in Macro-
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located within a distance of 6 Å from the ligand, while the
remaining residues were conformationally frozen. The following
parameters were employed for the conformational search: number
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100; energy window for saving structures ) 1000.0 kJ/mol. The
calculations were conducted with the MMFF force field,45 using
water as implicit solvent (GB/SA model as implemented in
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energy minimizations the Polak-Ribier conjugate gradient was used
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Acknowledgment. Dr. Pedro Besada and Dr. Dae Hong Shin
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Uridine 5′-Diphosphate Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5543
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